1. Field of the Invention
The present invention relates generally to human recombinant and synthetic granulocyte-macrophage colony stimulating factor (GM-CSF) low-affinity receptor proteins, antibodies and to derivatives thereof. These proteins are useful inter alia in the preparation of therapeutics and diagnostics and in the generation of agonist and antagonist compounds with respect to the binding GM-CSF to its receptor, which are also part of the present invention, including diagnostic and pharmaceutic compositions thereof. The present invention also provides diagnostic and therapeutic methods using the above proteins and compositions.
2. Related Art
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein growth and differentiation factor which regulates the proliferation, differentiation and functional activity of cells of the neutrophil, eosinophil and monocyte/macrophage series (reviewed in Metcalf, D., The Hemopoietic Colony Stimulating Factors, Elsevier, Amsterdam (I1984) and Gough, N. M. and Nicola, N. A., in Colony-Stimulating Factors: Molecular and Cellular Biology, Hexter, T. M. et al., eds., Marcel Dekker, New York, pp. 111-153 (1989). Molecular clones encoding murine (Gough, N. M. et al., Nature 309:763-767 (1984)) and human (Wong, G. G. et al., Science 228:810 (1985)) GM-CSF have been isolated, and recombinant protein tested in animal model systems (Metcalf, D. et al., Exp. Hematol. 15:1-9 (1987), Donohue, R. E. et al., Nature 321:827-875 (1986)) and in phase I/II clinical trials in patients with a variety of hemopoietic disorders (reviewed in Morstyn, G. et al., Trends Pharmacol. Sci. 10:154-159 (1989)). In both animal experiments and clinical trials, GM-CSF has been found to elevate circulating levels of monocytes, neutrophils and eosinophils; to enhance the functional capacities of the circulating cells; and to enhance the rate of hemopoietic recovery following chemotherapy and/or bone marrow transplantation (Gough, N. M. and Nicola, N. A., in Colony-Stimulating Factors: Molecular and Cellular Biology, Hexter, T. M. et al., eds., Marcel Dekker, New York, pp. 111-153 (1989); Morstyn, G. et al., Trends Pharmacol. Sci. 10:154-159 (1989)).
In both murine and human systems, autoradiographic analyses have indicated that GM-CSF receptors are present in low numbers (a few hundred per cell) on cells within the monocyte, neutrophil and eosinophil lineages (Nicola, N. A., Immunol. Today 8:134-139 (1987); DiPersio, J. et al., J. Biol. Chem. 263:1834-1840 (1988)). However, functional GM-CSF receptors have also been detected on non-hemopoietic cells, including endothelial cells (Bussolino, F. et al, Nature 337:471-473 (1989)), small-cell lung carcinoma cell lines and SV40-transformed simian COS cells (Cocita Baldwin, G. et al., Blood 73:1033-1037 (1989)).
In the murine system, Walker, F. and Burgess, A. W., EMBO J 4:933-939 (1985) detected both high-affinity (K.sub.D about 30 pM) and low-affinity (K.sub.D about 1 nM) receptors, while Park, L. L. et al., J. Biol. Chem. 261:4177-4183 (1986) detected a single receptor class of K.sub.D 1-3 nM. In the human system, only high-affinity receptors have been described on hemopoietic and endothelial cells (K.sub.D about 30 pM) (Gasson, J. C. et al., Proc. Natl. Acad. Sci. (USA) 83:669-673 (1986); Bussolino, F. et al., Nature 337:471-473 (1989); Park, L. L. et al., J. Exp. Med. 164:251-262 (1986)), but a receptor of lower affinity has been described on COS cells (Cocita Baldwin, G. et al., Blood 73:1033-1037 (1989)). In the murine system, the GM-CSF receptors recognize only GM-CSF, although they can be indirectly down-modulated by interleukin-3 and other agents (Walker, F. et al., Cell 45:269-276 (1985); Nicola, N. A., Immunol. Today 8:134-139 (1987)).
Because most of the biological effects of GM-CSF are observed at picomolar concentrations (Metcalf, D., The Hemopoietic Colony Stimulating Factors, Elsevier, Amsterdam (1984)), and because we have found that high-affinity receptors are preferentially internalized (Gough, N. M. and Nicola, N. A., in Colony-Stimulating Factors: Molecular and Cellular Biology, Hexter, T. M. et al., eds., Marcel Dekker, New York, pp. 111-153 (1989)), it is not clear whether low-affinity receptors are biologically functional in the murine system at 37.degree. C. (Walker, F. et al., Cell 45:269-276 (1985)) and in the human system at 4.degree. C. or 37.degree. C. (Elliot, M. J. et al., Blood 74:2349-2359 (1989); Lopez, A. F. et al., Proc. Natl. Acad. Sci. (USA) 86:7022-7026 (1989); Park, L. S. et al., J. Biol. Chem. 264:5420-5427 (1989)), Multi-CSF (interleukin-3) can down-modulate GM-CSF receptors on some types of hemopoietic cells, but it is not clear whether this is mediated by different receptor subclasses or by receptor-receptor interactions (Gearing et al., 1989; Elliot, M. J. et al., Blood 74:2349-2359 (1989); Lopez, A. F. et al., Proc. Natl. Acad. Sci. (USA) 86:7022-7026 (1989)).
GM-CSF was originally defined by its ability to stimulate the proliferation of granulocyte/macrophage progenitor cells, but more recently it has become apparent that it can also stimulate the proliferation of progenitor cells of other hemopoietic lineages (Metcalf, D. et al., Blood 55:138-147 (1980)) and cells of non-hemopoietic origin. The latter include human bone marrow fibroblasts, osteogenic sarcoma cell lines and a breast carcinoma cell line (Dedhar, S. et al., Proc. Natl. Acad. Sci. (USA) 85:9253-9257 (1988)), human small cell carcinoma cell lines (Cocita Baldwin, G. et al., Blood 73:1033-1037 (1989)), human endothelial cells (Bussolino, F. et al., Nature 337:471-473 (1989)) and human placental cells (Wegmann, T. G. et al., Transplant. Proc. 21:566-568 (1989)). In addition, GM-CSF stimulates human endothelial cell migration (Bussolino, F. et al., Nature 337:471-473 (1989)), the proliferation and function of human osteoblast-like cells in vitro (Evans, D. B. et al., Biochem. Biophys. Res. Commun. 160:588-595 (1989)) and improves the growth of murine placental cells in vivo (Wegmann, T. G. etal., Transplant. Proc. 21:566-568 (1989)).
In view of these biological results, it is apparent that the GM-CSF receptors detected on these cells are functional, despite the fact that only high affinity receptors were detected on the endothelial cells, whereas only low affinity receptors were detected on- the fibroblasts and placental membranes.
On hemopoietic cells, low affinity hGM-CSF receptors were distinguished by a rapid rate of ligand dissociation and poor internalization at 37.degree. C., whereas high affinity receptors displayed a much slower rate of ligand dissociation, and were efficiently internalized. In addition to this complexity, two types of high affinity hGM-CSF receptor have been described on some but not all normal hemopoietic cells and cell lines. One type recognizes only hGM-CSF and is the only type of GM-CSF receptor on human neutrophils, while the other type apparently recognizes hGM-C-SF and h-IL-3 with nearly equal affinity, and represents 80% of GM-CSF receptors on eosinophils (Lopez, A. F. et al., Proc. Natl. Acad. Sci. (USA) 86:7022-7026 (1989)). Reciprocally, IL-3-specific or cross-reactive receptors have also been described (Park, L. S. et al., J. Biol. Chem. 264:5420-5427 (1989)).
Finally, cross-linking experiments have suggested a molecular weight for the murine GM-CSF receptor of 51,000 (Walker, F. and Burgess, A. W., EMBO J. 4:933-939 (1985)) or 130,000 (Park, L. L. et al., J. Biol. Chem. 261:4177-4183 (1986)), while the human receptor molecular weight has been estimated at 84,000 (DiPersio, J. et al., J. Biol. Chem. 263:1834-1840 (1988)).
Although some of the properties of GM-CSF receptor have been deduced, the receptor has not hitherto been isolated or purified.